Driegen Siska, Ferreira Rita, van Zon Arend, Strouboulis John, Jaegle Martine, Grosveld Frank, Philipsen Sjaak, Meijer Dies
Department of Cell Biology and Genetics, Erasmus MC, Rotterdam, The Netherlands.
Transgenic Res. 2005 Aug;14(4):477-82. doi: 10.1007/s11248-005-7220-2.
The remarkable high affinity (Kd approximately 10(-15) M) of avidin/streptavidin for biotin has been extensively exploited in purification methodologies. Recently a small peptide sequence (Avi-tag) has been defined that can be specifically and efficiently biotinylated by the bacterial BirA biotin ligase. Fusion of this small peptide sequence to a protein of interest and co-expression with the BirA gene in mammalian cells allowed purification of the biotinylated protein together with its associated proteins and other molecules. Ideally, one would like to apply these technologies to purify tagged proteins directly from mouse tissues. To make this approach feasible for a large variety of proteins we developed a mouse strain that expresses the BirA gene ubiquitously by inserting it in the ROSA26 locus. We demonstrate that the BirA protein is indeed expressed in all tissues tested. In order to demonstrate functionality we show that it biotinylates the transgene-encoded Avi-tagged Gata1 and Oct6 transcription factors in erythroid cells of the foetal liver and Schwann cells of the peripheral nerve respectively. Therefore, this mouse can be crossed to any transgenic mouse to obtain efficient biotinylation of an Avi-tagged protein for the purpose of protein (complex) purification.
抗生物素蛋白/链霉抗生物素蛋白与生物素具有极高的亲和力（解离常数Kd约为10^(-15) M），这一特性已在多种纯化方法中得到广泛应用。最近，人们确定了一段小肽序列（Avi标签），它可被细菌BirA生物素连接酶特异性且高效地生物素化。将此小肽序列与目标蛋白融合，并在哺乳动物细胞中与BirA基因共表达，可实现生物素化蛋白及其相关蛋白和其他分子的纯化。理想情况下，人们希望应用这些技术直接从小鼠组织中纯化带标签的蛋白。为使该方法适用于多种蛋白，我们构建了一种小鼠品系，通过将BirA基因插入ROSA26位点使其在全身表达。我们证明BirA蛋白确实在所有检测的组织中都有表达。为证明其功能，我们分别展示了它能在胎肝的红细胞和外周神经的施万细胞中对转基因编码的带有Avi标签的Gata1和Oct6转录因子进行生物素化。因此，这种小鼠可与任何转基因小鼠杂交，以实现对带有Avi标签的蛋白进行高效生物素化，用于蛋白（复合物）纯化。
